The expression of exogenous DNA in eukaryotic cells permits the study of a broad array of biological topics ranging from the regulation of gene expression to the treatment of disease by gene transfer-based therapies. A number of methods for gene transfer into mammalian cells have evolved. These include in vivo and in vitro infection with cloned retroviral vectors (Shimotohno, K., and Temin, H. M. (1981) Cell 26:67-77; Cone, R. D., and Mulligan, R. C. (1984) Proc. Natl. Acad. Sci. USA 81:6349-6353; Dubensky, T. W., Campbell, B. A., and Villareal, L. P. (1984) Proc. Natl. Acad. Sci. USA 81:7529-7533; Seeger, C., Ganem, D. and Varmus, H. E. (1984) Proc. Natl. Acad. Sci. USA 81:5849-5852), coprecipitation of DNA with calcium phosphate (Chu, G., and Sharp, P. (1981) Gene 13:197-202; Benvenisty, N., and Reshef, L. (1986) Proc. Natl. Acad. Sci. USA 83:9551-9555), encapsulation of DNA in liposomes (Felgner, P. L., and Ringold, G. M. (1989) Nature 337:387-388; Kaneda, Y., Iwai, K., and Uchida, T. (1989) Science 243:375-378), direct injection of plasmid DNA (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A., and Felgner, P. L. (1990) Science 247:1465-1468), DEAE-dextran (McCutchan, J. H., and Pagano, J. S. (1968) J. Natl. Cancer Inst. 41:351-357), electroporation (Neumann, E., Schaefer-Ridder, M., Wang, Y., and Hofschneider, P. H. (1982) EMBO J. 1:841-845; Cann, A. J., Koyanagi, Y., and Chen, I. S. Y. (1988) Oncogene 3:123-128), and DNA-coated particle bombardment of cells and tissues (Yang, N-S., Burkholder, J., Roberts, B., Martinell, B., and McCabe, D. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572).
Although transfection of numerous cell types with an exogenous nucleic acid molecule containing a gene results in efficient expression of the exogenous gene, primary T lymphocytes, e.g. peripheral blood T lymphocytes obtained from an individual, have been found to be refractory to transfection and expression of exogenous DNA. Primary T lymphocytes also have been found to be refractory to expression of the introduced nucleic acid when first stimulated to proliferate. Thus, a system that allows for efficient introduction of exogenous DNA into primary T cells and expression of the exogenous DNA in the T cell is still needed.
The present invention provides an improved method for transfecting T cells with a nucleic acid molecule containing a gene such that expression of the gene in the T cells is enhanced as compared to classic transfection techniques. The method of the invention is particularly useful for transfecting primary T cells which are refractory to classical transfection techniques. The method involves contacting a proliferating T cell with one or more agents which stimulate the proliferating T cell prior to introducing the nucleic acid molecule into the T cell. In one embodiment of the invention, the T cell is stimulated with a combination of a first agent which provides a primary activation signal to the T cell and a second agent which provides a costimulatory signal. The method of the invention has numerous applications, in particular for gene therapy.